Peptides for detection of antibody to Anaplasma phagocytophilum

ABSTRACT

The invention provides compositions and methods for the detection and quantification of  A. phagocytophilum  (formerly known as  Ehrlichia equi ) antibodies and antibody fragments.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 11/033,209 filed Jan.11, 2005 (now U.S. Pat. No. 7,439,321), which is a divisionalapplication of U.S. Ser. No. 10/404,626, filed Apr. 1, 2003 (now U.S.Pat. No. 6,964,855), which is a continuation-in-part of U.S. Ser. No.10/121,799 filed Apr. 12, 2002 (now abandoned), all of which areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The invention relates to methods and compositions for the detection andquantification of Anaplasma phagocytophilum antibodies and antibodyfragments. A. phagocytophilum is formerly known as Ehrlichia equi.

BACKGROUND OF THE INVENTION

Granulocytic ehrlichiosis occurs in mammals such as humans, horses, dogsand cats and is caused by infection of granulocytic cells with thetick-borne agent Anaplasma phagocytophilum (formerly known as Ehrlichiaequi). Frequently reported symptoms of granulocytic ehrlichiosis inhumans are leukopenia and thrombocytopenia. Common clinical signs indogs and horses are fever and anexoria.

Indirect immunofluorescence assays (IFA) and enzyme-linked immunosorbentassays (ELISA) are frequently used as aids in the diagnosis of diseasescaused by A. phagocytophilum by measuring the binding of antibody from apatient's blood or serum to infected cells, cell lysates or purifiedehrlichial proteins. However, these assays are severely limited inusefulness because of sensitivity and specificity issues directlyrelated to the impure nature of the antigen used in these tests. Highlypurified reagents are needed to construct more accurate assays. Thisinvention discloses specific synthetic peptide sequences derived from A.phagocytophilum that can be used in place of partially purifiedproteins, infected cells or cell lysates.

SUMMARY OF THE INVENTION

It is an object of the invention to provide methods and compositions forthe detection and quantification of A. phagocytophilum antibodies andantibody fragments. This and other objects of the invention are providedby one or more of the embodiments described below.

One embodiment of the invention provides a composition of matterconsisting essentially of an isolated polypeptide shown in SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:3. The composition can comprise a carrier. Theisolated polypeptide of the composition can be conjugated to bovineserum albumin. The polypeptide of the composition can consistessentially of a fragment of at least about 5 contiguous amino acids ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. The invention also comprisesan isolated polynucleotide encoding the isolated polypeptide of thecomposition.

Another embodiment of the invention provides a method of detectingantibodies specific for A. phagocytophilum. The method comprisescontacting a polypeptide shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,or a combination of two or three polypeptides SEQ ID NO:1, SEQ ID NO:2,and SEQ ID NO:3 with a test sample suspected of comprising antibodiesthat are specific for A. phagocytophilum, under conditions that allowpolypeptide/antibody complexes to form. The polypeptide can be attachedto a substrate and can be in a multimeric form. The test sample can abiological sample obtained from a mammal, such as a human, cat, horse ordog. Polypeptide/antibody complexes are detected. The detection ofpolypeptide/antibody complexes is an indication that antibodies specificfor A. phagocytophilum are present in the test sample. Thepolypeptide/antibody complexes can be contacted with an indicatorreagent comprising a signal generating compound prior to the detectionstep. The antibodies can be antibody fragments. The amount of antibodyin a test sample can be determined using this method. The method cancomprise an assay selected from the group of assays consisting of areversible flow chromatographic binding assay, an enzyme linkedimmunosorbent assay, a radioimmunoassay, a hemagglutination assay awestern blot assay, a fluorescence polarization immunoassay and anindirect immunofluorescence assay.

Still another embodiment of the invention comprises an article ofmanufacture comprising packaging material and, contained within thepackaging material, a polypeptide shown in SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3, or a combination of two or three polypeptides shown in SEQID NO:1, SEQ ID NO:2, and SEQ ID NO:3. The packaging material cancomprise a label that indicates that the one or more polypeptides can beused for the identification of A. phagocytophilum infection in a mammal.

Even another embodiment of the invention provides a method of diagnosingan A. phagocytophilum infection in a mammal. The method comprisesobtaining a biological sample from a mammal suspected of having an A.phagocytophilum infection and contacting a polypeptide shown in SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3 or a combination of two or threepolypeptides SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, with thebiological sample under conditions that allow polypeptide/antibodycomplexes to form. The polypeptide/antibody complexes are detected. Thedetection of polypeptide/antibody complexes is an indication that themammal has an A. phagocytophilum infection. The polypeptide/antibodycomplexes can be contacted with an indicator reagent comprising a signalgenerating compound prior to the detection step. The mammal can be ahuman, cat, horse or dog.

Another embodiment of the invention provides an antibody thatspecifically binds to at least one epitope of an A. phagocytophilumpolypeptide, wherein said polypeptide is SEQ ID NO:1, SEQ ID NO:2 or SEQID NO:3. The antibody can be a monoclonal antibody.

The invention therefore provides methods and compositions that can beused to detect A. phagocytophilum antibodies and antibody fragments withimproved sensitivity and specificity.

DETAILED DESCRIPTION OF THE INVENTION

Immunodominant regions of a P30 protein of E. canis have previously beenidentified using phage display technology. See U.S. patent applicationSer. No. 09/765,736 filed Jan. 18, 2001. The identified sequencesexhibited strong homology to sequences for outer membrane proteins ofseveral isolates of Ehrlichia canis. Synthetic peptides corresponding tosequences from homologous regions of several outer membrane proteinshave been synthesized and used in diagnostic assays to detect antibodiesand antibody fragments to E. canis.

A. phagocytophilum and E. canis are different species of relatedorganisms that are classified within different serotypes of theEhrlichia group. Polypeptide sequences of A. phagocytophilum wereexamined to identify immunodominant regions. Immunodominant sequencesderived from an A. phagocytophilum membrane protein, GE E8 msp-2, wereidentified by comparison to E. canis immunodominant polypeptides (Murphyet al., Infection and Immunity, vol. 66(8), pp. 3711-3718 (1998)). Thefollowing sequences, which correspond to amino acids numbers 74 to 99,62 to 92 and 20-39 of an A. phagocytophilum membrane protein, wereidentified and used as a basis to synthesize three synthetic peptides:

Peptide I. Amino Acid Nos. 74-99 (SEQ ID NO: 1) K D G K S V K L E S H KF D W N T P D P R I G F K D Peptide II. Amino Acid Nos. 65-92 (SEQ IDNO: 2) E T K A V Y P Y L K D G K S V K L E S H K F D W N T P D PeptideIII. Amino Acid Nos. 120-139 (SEQ ID NO: 3) L E I G Y E R F K T K G I RD S G S K E

A. phagocytophilum Polypeptides

In one embodiment of the invention, a polypeptide or fragment thereof issubstantially pure. Substantially pure means that a polypeptide of theinvention is substantially free from other biological molecules. Asubstantially pure polypeptide is at least 75%, 80%, 90%, 95%, 97%, 99%or 100% pure by dry weight. Purity can be measured by a method such ascolumn chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

Polypeptides of the invention can also comprise fragments of thepolypeptides shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. Forexample, fragments of polypeptides can comprise at least about 5, 6, 8,10, 12, 15, 18, 20, 22, 24, or 26 contiguous amino acids of thepolypeptides shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

Polypeptides of the invention can also comprise conservative variants ofthe polypeptides shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. Aconservative variant is a polypeptide that differs from SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3, or a fragment thereof, only in conservativesubstitutions, such that the antigenic properties of the polypeptide aresubstantially the same as the original polypeptide. Conservativevariants can generally be identified by modifying a polypeptide sequenceof the invention and evaluating the antigenic activity of the modifiedpolypeptide using, for example, an immunohistochemical assay, anenzyme-linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), ora western blot assay. A variant, including a conservative variant and anantigenically active variant will bind to an anti-A. phagocytophilumantibody or antibody fragment with substantially the same bindingspecificity of a polypeptide shown in SEQ ID NO:1, SEQ ID NO:2, or SEQID NO:3. “Binding specificity” or “specifically binds” means that apolypeptide will substantially recognize and bind to an anti-A.phagocytophilum polyclonal or monoclonal antibody or antibody fragment(e.g., a Fv, single chain Fv, Fab′, or F(ab′)₂ fragment), but does notsubstantially bind to other molecules in a test sample. Specific bindingcan be tested using, e.g, an immunohistochemical assay, an enzyme-linkedimmunosorbant assay (ELISA), a radioimmunoassay (RIA), or a western blotassay. Polypeptides of the invention can comprise up to about 1, 2, 3,5, 6, 10, or 15 conservative amino acid substitutions.

A conservative substitution is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. In general, the following groups of amino acids representconservative changes: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr;(2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg,his; and (5) phe, tyr, trp, his. That is, amino acids within each of thegroups can be substituted for another amino acid of the same group.

A polypeptide of the invention or fragments thereof can differ from thecorresponding sequence in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 andyet retain substantially the same antigenic activity of the originalpolypeptide or fragment. This is an antigenically active variant or anantigenically active fragment. A conservative variant is one type of anantigenically active variant or fragment. Naturally occurringantigenically active variants, such as allelic variants andnon-naturally occurring antigenically active variants, are included inthe invention and can be produced by, for example, mutagenesistechniques or by direct synthesis.

An antigenically-active variant differs by about, for example, 1, 2, 3,5, 6, 10, 15 or 20 amino acid residues from a polypeptide shown in SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3 or a fragment thereof. Where thiscomparison requires alignment the sequences are aligned for maximumhomology. Deletions, insertions, substitutions, repeats, inversions ormismatches are considered differences. The differences are, preferably,differences or changes at a non-essential residue or a conservativesubstitution. The site of variation can occur anywhere in thepolypeptide, as long as the resulting variant polypeptide isantigenicity substantially similar to a polypeptide shown in SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3.

A polypeptide is an antigenically active variant or antigenically activefragment if it reacts substantially the same as a polypeptide shown inSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 in an assay such as animmunohistochemical assay, an ELISA, an RIA, an IFA, or a western blotassay, e.g. has about 90-110% of the activity of the originalpolypeptide. In one embodiment, the assay is a competition assay whereinthe antigenically active variant polypeptide or fragment is capable ofreducing binding of a polypeptide shown in SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3 to a corresponding reactive antigen or antibody by about 90,95, 99, or 100%.

Therefore, the invention provides antigenically active variantpolypeptides that can be at least 85% identical, more preferably atleast 90% identical, and still more preferably at least 95%, 96%, 97%,98%, or 99% identical to a polypeptide sequence shown in SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:3.

Identity or identical means amino acid sequence similarity and has anart recognized meaning. Sequences with identity share identical orsimilar amino acids, where similar amino acids are preferably conservedamino acids. Thus, a candidate sequence sharing 90% amino acid sequenceidentity with a reference sequence (i.e., SEQ ID NO:1) requires that,following alignment of the candidate sequence with the referencesequence, 90% of the amino acids in the candidate sequence are identicalto the corresponding amino acids in the reference sequence, and/orconstitute conservative amino acid changes.

Sequences are aligned for identity calculations using a mathematicalalgorithm, such as the algorithm of Karlin and Altschul (1990) Proc.Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the XBLAST programs of Altschul et al. (1990) J. Mol.Biol. 215:403-410. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceswith identity to the polypeptides of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST) can be used. Internal gaps andamino acid insertions in the candidate sequence as aligned are ignoredwhen making the identity calculation.

Methods of introducing a mutation into amino acids of a protein is wellknown to those skilled in the art. See, e.g., Ausubel (ed.), CurrentProtocols in Molecular Biology, John Wiley and Sons, Inc. (1994); T.Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989).Mutations can also be introduced using commercially available kits suchas “QUIKCHANGE™ Site-Directed Mutagenesis Kit” (Stratagene). Thegeneration of a polypeptide antigenically substantially equivalent to apolypeptide shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 byreplacing an amino acid that does not influence the antigenicity of apolypeptide of the invention can be accomplished by one skilled in theart.

Polypeptides of the invention comprise at least one epitope that isrecognized by an anti-A. phagocytophilum antibody or fragment. Anepitope is an antigenic determinant of a polypeptide. An epitope can bea linear, sequential, or conformational epitope. Epitopes within apolypeptide of the invention can be identified by several methods. Seee.g., U.S. Pat. No. 4,554,101; Jameson & Wolf, CABIOS 4:181-186 (1988).For example, a polypeptide of the invention can be isolated andscreened. A series of short peptides, which together span the entirepolypeptide sequence, can be prepared by proteolytic cleavage. Bystarting with, for example, 20-mer polypeptide fragments, each fragmentcan be tested for the presence of epitopes recognized in, for example,an enzyme-linked immunosorbent assay (ELISA). In an ELISA assay apolypeptide, such as a 20-mer polypeptide fragment, is attached to asolid support, such as the wells of a plastic multi-well plate. Apopulation of antibodies or antibody fragments are labeled, added to thesolid support and allowed to bind to the unlabeled antigen, underconditions where non-specific adsorbtion is blocked, and any unboundantibody and other proteins are washed away. Antibody binding isdetected by, for example, a reaction that converts a colorless indicatorreagent into a colored reaction product. Progressively smaller andoverlapping fragments can then be tested from an identified 20-mer tomap the epitope of interest.

Preferably, a polypeptide of the invention is synthesized usingconventional peptide synthesizers, which are well known in the art. Apolypeptide of the invention can also be produced recombinantly. Apolynucleotide encoding an A. phagocytophilum polypeptide can beintroduced into an expression vector that can be expressed in a suitableexpression system using techniques well known in the art. A variety ofbacterial, yeast, plant, mammalian, and insect expression systems areavailable in the art and any such expression system can be used.Optionally, a polynucleotide encoding an A. phagocytophilum polypeptidecan be translated in a cell-free translation system.

A polypeptide of the invention can be produced as a fusion protein thatcontains other amino acid sequences, such as amino acid linkers orsignal sequences, as well as ligands useful in protein purification,such as glutathione-S-transferase, histidine tag, and staphylococcalprotein A. More than one polypeptide of the invention can be present ina fusion protein. The polypeptide can be fused to the N-terminus orC-terminus of a polypeptide of the invention.

A polypeptide of the invention can be synthesized such that it comprisesseveral repeated A. phagocytophilum polypeptides. This is a multimericpolypeptide. These repeated polypeptides can comprise one specificpolypeptide, e.g. the polypeptide shown in SEQ ID NO:1, repeated 2 ormore times. Alternatively, the repeated polypeptides can comprise one ormore copies of an A. phagocytophilum polypeptide. For example, suchpolypeptides can comprise one or more copies of an A. phagocytophilumpolypeptide shown in SEQ ID NO:1 along with one or more copies of an A.phagocytophilum polypeptide shown in SEQ ID NO:2 and/or SEQ ID NO:3. Apolypeptide of the invention can be combined or synthesized with one ormore non-A. phagocytophilum polypeptides, fragments of polypeptides, orfull-length polypeptides.

A polypeptide of the invention can be combined with a carrier. A carrieris a vehicle for a polypeptide of the invention. Carriers include, forexample, excipients, diluents, adjuvants, and stabilizers. Examples ofsuch stabilizers are proteins such as serum albumins and gelatin;saccharides such as glucose, sucrose, lactose, maltose, trehalose,sorbitol, maltitol, mannitol and lactitol; and buffers which are mainlycomposed of phosphate or succinate.

A. phagocytophilum Polynucleotides

Polynucleotides of the invention contain less than an entire microbialgenome and can be RNA or single- or double-stranded DNA. Preferably, thepolynucleotides are purified free of other components, such as proteinsand lipids. The polynucleotides of the invention encode the polypeptidesdescribed above. Polynucleotides of the invention can also compriseother nucleotide sequences, such as sequences coding for linkers, signalsequences, heterologous signal sequences, TMR stop transfer sequences,transmembrane domains, or ligands useful in protein purification such asglutathione-S-transferase, histidine tag, and staphylococcal protein A.

Polynucleotides can be synthesized in the laboratory, for example, usingan automatic synthesizer. An amplification method such as PCR can beused to amplify polynucleotides from either genomic DNA or cDNA encodingthe polypeptides.

If desired, polynucleotides can be cloned into an expression vectorcomprising, for example, promoters, enhancers, or other regulatoryelements that drive expression of the polynucleotides of the inventionin host cells. An expression vector can be, for example, a plasmid, suchas pBR322, pUC, or ColE1, or an adenovirus vector, such as an adenovirusType 2 vector or Type 5 vector.

A vector comprising a polynucleotide of the invention can be transformedinto, for example, bacterial, yeast, insect, or mammalian cells so thatthe polypeptides of the invention can be expressed in and isolated fromcell culture. Any techniques that are known and available in the art canbe used to introduce polynucleotides into the host cells. These include,but are not limited to, transfection with naked or encapsulated nucleicacids, cellular fusion, protoplast fusion, viral infection, andelectroporation.

Polynucleotides of the invention can be used to produce polypeptides ofthe invention and, for example, as probes or primers to detect thepresence of A. phagocytophilum polynucleotides in a sample, such as abiological sample. The ability of such probes to specifically hybridizeto A. phagocytophilum polynucleotide sequences will enable them to be ofuse in detecting the presence of complementary sequences in a givensample.

Methods of Detection

The methods of the invention can be used to detect antibodies orantibody fragments specific for A. phagocytophilum in a test sample,such as a biological sample, an environmental sample, or a laboratorysample. A biological sample can include, for example, sera, blood,cells, plasma, or tissue from a mammal such as a horse, cat, dog orhuman. The test sample can be untreated, precipitated, fractionated,separated, diluted, concentrated, or purified before combining with apolypeptide of the invention.

The methods comprise contacting a polypeptide of the invention with atest sample under conditions that allow a polypeptide/antibody complexto form. That is, a polypeptide of the invention specifically binds toan antibody specific for A. phagocytophilum located in the sample. Theformation of a complex between the polypeptide and anti-A.phagocytophilum antibodies in the sample is detected. In one embodimentof the invention, the polypeptide/antibody complex is detected when anindicator reagent, such as an enzyme, which is bound to the antibody,catalyzes a detectable reaction. Optionally, an indicator reagentcomprising a signal generating compound can be applied to thepolypeptide/antibody complex under conditions that allow formation of apolypeptide/antibody/indicator complex. Thepolypeptide/antibody/indicator complex is detected. Optionally, thepolypeptide or antibody can be labeled with an indicator reagent priorto the formation of a polypeptide/antibody complex. The method canoptionally comprise a positive or negative control.

Assays of the invention include, but are not limited to those based oncompetition, direct reaction or sandwich-type assays. Assays can usesolid phases or substrates or can be performed by immunoprecipitation orany other methods that do not utilize solid phases. Where a solid phaseor substrate is used, a polypeptide of the invention is directly orindirectly attached to a solid support or a substrate such as amicrotiter well, magnetic bead, non-magnetic bead, column, matrix,membrane, fibrous mat composed of synthetic or natural fibers (e.g.,glass or cellulose-based materials or thermoplastic polymers, such as,polyethylene, polypropylene, or polyester), sintered structure composedof particulate materials (e.g., glass or various thermoplasticpolymers), or cast membrane film composed of nitrocellulose, nylon,polysulfone or the like (generally synthetic in nature). A preferredsubstrate is sintered, fine particles of polyethylene, commonly known asporous polyethylene, for example, 10-15 micron porous polyethylene fromChromex Corporation (Albuquerque, N. Mex.). All of these substratematerials can be used in suitable shapes, such as films, sheets, orplates, or they may be coated onto or bonded or laminated to appropriateinert carriers, such as paper, glass, plastic films, or fabrics.Suitable methods for immobilizing peptides on solid phases includeionic, hydrophobic, covalent interactions and the like.

Polypeptides of the invention can be used to detect anti-A.phagocytophilum antibodies or antibody fragments in assays including,but not limited to enzyme linked immunosorbent assay (ELISA), westernblot, IFA, radioimmunoassay (RIA), hemagglutination (HA), andfluorescence polarization immunoassay (FPIA). A preferred assay of theinvention is the reversible flow chromatographic binding assay, forexample a SNAP® assay. See U.S. Pat. No. 5,726,010.

In one type of assay format, one or more polypeptides can be coated on asolid phase or substrate. A test sample suspected of containing ananti-A. phagocytophilum antibody or fragment thereof is incubated withan indicator reagent comprising a signal generating compound conjugatedto an antibody or antibody fragment specific for A. phagocytophilum fora time and under conditions sufficient to form antigen/antibodycomplexes of either antibodies of the test sample to the polypeptides ofthe solid phase or the indicator reagent compound conjugated to anantibody specific for A. phagocytophilum to the polypeptides of thesolid phase. The reduction in binding of the indicator reagentconjugated to an anti-A. phagocytophilum antibody to the solid phase canbe quantitatively measured. A measurable reduction in the signalcompared to the signal generated from a confirmed negative A.phagocytophilum test sample indicates the presence of anti-A.phagocytophilum antibody in the test sample. This type of assay canquantitate the amount of anti-A. phagocytophilum antibodies in a testsample.

In another type of assay format, one or more polypeptides of theinvention are coated onto a support or substrate. A polypeptide of theinvention is conjugated to an indicator reagent and added to a testsample. This mixture is applied to the support or substrate. If A.phagocytophilum antibodies are present in the test sample they will bindthe polypeptide conjugated to an indicator reagent and to thepolypeptide immobilized on the support. Thepolypeptide/antibody/indicator complex can then be detected. This typeof assay can quantitate the amount of anti-A. phagocytophilum antibodiesin a test sample.

The formation of a polypeptide/antibody complex or apolypeptide/antibody/indicator complex can be detected by radiometric,colormetric, fluorometric, size-separation, or precipitation methods.Optionally, detection of a polypeptide/antibody complex is by theaddition of a secondary antibody that is coupled to an indicator reagentcomprising a signal generating compound. Indicator reagents comprisingsignal generating compounds (labels) associated with apolypeptide/antibody complex can be detected using the methods describedabove and include chromogenic agents, catalysts such as enzymes,fluorescent compounds such as fluorescein and rhodamine,chemiluminescent compounds such as dioxetanes, acridiniums,phenanthridiniums, ruthenium, and luminol, radioactive elements, directvisual labels, as well as cofactors, inhibitors, magnetic particles, andthe like. Examples of enzymes include alkaline phosphatase, horseradishperoxidase, beta-galactosidase, and the like. The selection of aparticular label is not critical, but it will be capable of producing asignal either by itself or in conjunction with one or more additionalsubstances.

Formation of the complex is indicative of the presence of anti-A.phagocytophilum antibodies in a test sample. Therefore, the methods ofthe invention can be used to diagnose A. phagocytophilum infection in apatient.

The methods of the invention can also indicate the amount or quantity ofanti-A. phagocytophilum antibodies in a test sample. With many indicatorreagents, such as enzymes, the amount of antibody present isproportional to the signal generated. Depending upon the type of testsample, it can be diluted with a suitable buffer reagent, concentrated,or contacted with a solid phase without any manipulation. For example,it usually is preferred to test serum or plasma samples that previouslyhave been diluted, or concentrate specimens such as urine, in order todetermine the presence and/or amount of antibody present.

The invention further comprises assay kits (e.g., articles ofmanufacture) for detecting anti-A. phagocytophilum antibodies orantibody fragments in a sample. A kit or article of manufacturecomprises one or more polypeptides of the invention and means fordetermining binding of the polypeptide to A. phagocytophilum antibodiesor antibody fragments in the sample. A kit can comprise a devicecontaining one or more polypeptides of the invention and instructionsfor use of the one or more polypeptides for the identification of an A.phagocytophilum infection in a mammal. The kit can also comprisepackaging material comprising a label that indicates that the one ormore polypeptides of the kit can be used for the identification of A.phagocytophilum infection. Other components such as buffers, controls,and the like, known to those of ordinary skill in art, can be includedin such test kits. The polypeptides, assays, and kits of the inventionare useful, for example, in the diagnosis of individual cases of A.phagocytophilum infection in a patient, as well as epidemiologicalstudies of A. phagocytophilum outbreaks.

Polypeptides and assays of the invention can be combined with otherpolypeptides or assays to detect the presence of A. phagocytophilumalong with other organisms. For example, polypeptides and assays of theinvention can be combined with reagents that detect heartworm and/orBorrelia burgdorferi.

Monoclonal Antibodies

The polypeptides of the invention can also be used to develop monoclonaland/or polyclonal antibodies that specifically bind to an immunologicalepitope of A. phagocytophilum present in the polypeptides of theinvention.

The antibodies or fragments thereof can be employed in assay systems,such as a reversible flow chromatographic binding assay, enzyme linkedimmunosorbent assay, western blot assay, or indirect immunofluorescenceassay, to determine the presence, if any, of A. phagocytophilumpolypeptides or antibodies in a test sample. In addition, theseantibodies, in particular monoclonal antibodies, can be bound tomatrices similar to CNBr-activated Sepharose and used for the affinitypurification of specific A. phagocytophilum proteins from, for example,cell cultures or blood serum, such as to purify recombinant and nativeA. phagocytophilum antigens and proteins. The monoclonal antibodies ofthe invention can also be used for the generation of chimeric antibodiesfor therapeutic use, or other similar applications.

Monoclonal antibodies directed against A. phagocytophilum epitopes canbe produced by one skilled in the art. The general methodology forproducing such antibodies is well-known and has been described in, forexample, Kohler and Milstein, Nature 256:494 (1975) and reviewed in J.G. R. Hurrel, ed., Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press Inc., Boca Raton, Fla. (1982), as well as thattaught by L. T. Mimms et al., Virology 176:604-619 (1990). Immortalantibody-producing cell lines can be created by cell fusion, and also byother techniques such as direct transformation of B lymphocytes withoncogenic DNA, or transfection with Epstein-Barr virus.

The following are provided for exemplification purposes only and are notintended to limit the scope of the invention described in broad termsabove. All references cited in this disclosure are incorporated hereinby reference.

EXAMPLES Example 1 Canine

Three A. phagocytophilum antibody positive and three A. phagocytophilumantibody negative control canine samples (confirmed by Western blot)were obtained from the Connecticut Agricultural Experiment Station (NewHaven, Conn.). The positive samples were supplied with A.phagocytophilum antibody ELISA titers determined by the ConnecticutAgricultural Experiment Station using an A. phagocytophilum whole celllysate as an antigen source.

The A. phagocytophilum ELISA titers and results of the microtiter-platebased immunoassay were obtained using a mixture (50:50) of the syntheticpeptides shown in SEQ ID NO:1 and SEQ ID NO:2. Immunoassay syntheticpeptides were immobilized on microtiter wells. A dilution of the testsample was added to the microtiter well and unbound antibody was removedby washing. Antibody bound to the immobilized peptide was detected byreaction with an anti-species, in this case canine, horseradishperoxidase (HRPO) conjugate, washing and addition of a HRPO substrate.The optical density of individual microtiter wells was determined usinga microtiter plate reader. The results are shown in Table 1.

Example 2 Equine

Three A. phagocytophilum antibody positive and three A. phagocytophilumantibody negative control equine samples (confirmed by Western blot)were obtained from the Connecticut Agricultural Experiment Station. Thepositive samples were supplied with A. phagocytophilum antibody ELISAtiters determined by the Connecticut Agricultural Experiment Stationusing an A. phagocytophilum whole cell lysate as an antigen source. A.phagocytophilum ELISA titers and the results of a microtiter-plate basedimmunoassay were obtained using a mixture (50:50) of SEQ ID NO:1 and SEQID NO:2. The peptide-based assay was performed as described above usingan anti-equine:HRPO conjugate. The results are shown in Table 2.

Example 3 Feline

Three A. phagocytophilum antibody positive and two A. phagocytophilumantibody negative feline samples were obtained from Dr. Steve Levy, aConnecticut veterinarian. Samples were confirmed by animmunofluorescence assay (IFA) at North Carolina State University usingan A. phagocytophilum whole cell lysate as an antigen source.

The A. phagocytophilum titers determined by IFA and results of themicrotiter-plate based immunoassay were obtained using a mixture (50:50)of SEQ ID NO:1 and SEQ ID NO:2. The peptide-based assay was performed asdescribed above using an anti-feline:HRPO conjugate. The results areshown in Table 3.

Example 4 Canine (SEQ ID NO:3)

Three A. phagocytophilum antibody positive and three A. phagocytophilumantibody negative control canine samples, confirmed by IFA, wereobtained from Dr. Steve Levy.

Antibodies to SEQ ID NO:3 were determined by microtiter-plate basedimmunoassay. The synthetic peptide was immobilized on microtiter wells.A dilution of the test sample was added to the microtiter well andunbound antibody was removed by washing. Antibody bound to theimmobilized peptide was detected by reaction with an anti-species, inthis case canine, horseradish peroxidase (HRPO) conjugate, washing andaddition of a HRPO substrate. The optical density of individualmicrotiter wells was determined using a microtiter plate reader. Theresults are shown in Table 4.

Example 5 Equine (SEQ ID NO:3)

Three A. phagocytophilum antibody positive and three A. phagocytophilumantibody negative control equine samples confirmed by IFA were obtainedfrom Connecticut Veterinary Diagnostic Laboratory.

Antibodies to SEQ ID NO:3 were determined by microtiter-plate basedimmunoassay. The peptide-based assay was performed as described aboveusing an anti-equine:HRPO conjugate. The results are shown in Table 5.

Example 6 Feline (SEQ ID NO:3)

Three A. phagocytophilum antibody positive and three A. phagocytophilumantibody negative feline samples, confirmed by IFA, were obtained fromDr. Steve Levy.

Antibodies to SEQ ID NO:3 were determined by microtiter-plate basedimmunoassay. The peptide:based assay was performed as described aboveusing an anti-feline:HRPO conjugate. The results are shown in Table 6.

TABLE 1 Comparison of ELISA results using A. phagocytophilum whole celllysate as antigen source and A. phagocytophilum synthetic peptides. A.phagocytophilum Sample A. phagocytophilum Synthetic Peptides ID SpeciesELISA Titer/Result¹ ELISA OD/Result 2249 Canine  2560/Pos 0.068/Pos 2185Canine 20480/Pos 0.504/Pos 2292 Canine 10240/Pos 0.342/Pos WY05 CanineNeg 0.034/Neg WY023 Canine Neg 0.036/Neg WY013 Canine Neg 0.031/Neg¹Connecticut Agricultural Experiment Station

TABLE 2 Comparison of ELISA results using A. phagocytophilum whole celllysate as antigen source and A. phagocytophilum synthetic peptides. A.phagocytophilum Sample A. phagocytophilum Synthetic Peptides ID SpeciesELISA Titer/Result¹ ELISA OD/Result HO4a Equine 40960/Pos 0.261/Pos H46Equine  5120/Pos  0.48/Pos H22 Equine 20480/Pos 0.362/Pos Kent 29 EquineNeg 0.055/Neg Kent 26 Equine Neg 0.056/Neg Kent 30 Equine Neg 0.046/Neg¹Connecticut Agricultural Experiment Station

TABLE 3 Comparison of IFA results using A. phagocytophilum whole celllysate as antigen source and ELISA using A. phagocytophilum syntheticpeptides A. phagocytophilum A. phagocytophilum Sample Whole Cell LysateSynthetic Peptides ID Species IFA Titer/Result² ELISA OD/Result F8Feline 2048/Pos 0.678/Pos F15 Feline 2048/Pos 0.848/Pos F19 Feline 64/Pos 0.095/Pos F2 Feline Neg 0.036/Neg F3 Feline Neg 0.037/Neg ²NorthCarolina State University

TABLE 4 ELISA results using A. phagocytophilum synthetic peptide. A.phagocytophilum Synthetic Peptide Sample A. phagocytophilum SEQ ID NO: 3ID Species IFA ELISA OD/Result DP87 Canine Pos 0.742/Pos DP46 Canine Pos0.911/Pos DP20 Canine Pos 1.157/Pos DP81 Canine Neg 0.024/Neg DP88Canine Neg 0.006/Neg DP31 Canine Neg 0.031/Neg

TABLE 5 ELISA results using A. phagocytophilum synthetic peptide. A.phagocytophilum Synthetic Peptide Sample A. phagocytophilum SEQ ID NO: 3ID Species IFA ELISA OD/Result 42 Equine Pos 0.240/Pos 535 Equine Pos0.355/Pos 6 Equine Pos 0.369/Pos 98 Equine Neg 0.041/Neg 284 Equine Neg0.047/Neg 315 Equine Neg 0.048/Neg

TABLE 6 ELISA results using A. phagocytophilum synthetic peptide. A.phagocytophilum Synthetic Peptide Sample A. phagocytophilum SEQ ID NO: 3ID Species IFA ELISA OD/Result CP88 Feline Pos 0.250/Pos CP05 Feline Pos0.150/Pos CP02 Feline Pos 1.050/Pos CP27 Feline Neg 0.045/Neg CP42Feline Neg 0.043/Neg

1. A composition comprising a substantially purified polypeptideconsisting of SEQ ID NO:3.
 2. The composition of claim 1, furthercomprising a carrier.
 3. The composition of claim 1, wherein thesubstantially purified polypeptide is conjugated to bovine serumalbumin.
 4. An article of manufacture comprising a substantiallypurified polypeptide consisting of SEQ ID NO:3, and packaging materialtherefore.
 5. The article of manufacture of claim 4, wherein thepackaging material comprises a label that indicates that the one or morepolypeptides can be used for the identification of Anaplasmaphagocytophilum.
 6. A substantially purified polypeptide consisting ofSEQ ID NO:3.
 7. A fusion protein consisting of the polypeptide of claim6 and one or more non-Anaplasma phagocytophilum polypeptides.
 8. Thefusion protein of claim 7 wherein the one or more non-Anaplasmaphagocytophilum polypeptides are selected from the group consisting ofan amino acid linker, a signal sequence, a protein purification ligandor combinations thereof.
 9. A multimeric protein consisting of two ormore copies of the polypeptide of claim 6.